Roles of Essential Oils, Polyphenols, and Saponins of Medicinal Plants as Natural Additives and Anthelmintics in Ruminant Diets: A Systematic Review

Simple Summary Ruminant nutritionists have been challenged to improve animal production efficiently but at the same time produce healthy and environment friendly ruminant-derived food products. Recent studies on utilizing essential oils, polyphenols, and saponins of herbal plants show that these bioactive components can play important roles as alternative natural dietary additives and anthelmintics, in order to replace growth-promoting antibiotic and chemical anthelmintic treatments. Since the prohibition of using growth-promoting antibiotics and chemical anthelmintics, the global market has emphasized the use of natural feed additives and anthelmintics as alternatives for ruminants. This article presents the potentials and problems of using plant-based bioactive compounds for sustainable ruminant diets to support food safety and food security. Abstract Public awareness on health and safety issues in using antibiotics for livestock production has led many countries to ban the use of all growth-promoting antibiotics (GPA) for livestock feeding. The ban on the utilization of antibiotics in livestock, on the other hand, is an opportunity for researchers and livestock practitioners to develop alternative feed additives that are safe for both livestock and the consumers of animal derived foods. Many feed additives were developed from a number of plants that contain secondary metabolites, such as essential oils, polyphenols, and saponins. These secondary metabolites are extracted from various parts of many types of plants for their uses as feed additives and anthelmintics. Recent investigations on using essential oils, polyphenols, and saponins as dietary additives and anthelmintics demonstrate that they can increase not only the production and health of ruminants but also ensure the safety of the resulting foods. There are many publications on the advantageous impacts of dietary plant bioactive components on ruminants; however, a comprehensive review on individual bioactive constituents of each plant secondary metabolites along with their beneficial effects as feed additives and anthelmintics on ruminants is highly required. This current study reviewed the individual bioactive components of different plant secondary metabolites and their functions as additives and anthelmintics to improve ruminant production and health, with respect to safety, affordability and efficiency, using a systematic review procedure.


Introduction
Public awareness surrounding the health and safety issues of using antibiotics for livestock production, including ruminants, has led many countries such as the EU to Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) procedure [17], as explained in Figure 1.

Essential Oils Sources and Types
Essential oils (EO), recognzed as volatile oils, are commonly derived from edible, medicinal, herbal, or spice plants. The main plant tissues for EO deposition vary across the plants. They can be the leaves, flowers, stem, seeds, roots, rhizomes, or barks. The EO deposits are mostly extracted by using either steam distillation, hydro distillation, or organic solvent extractions [18]. The EO compounds are chemically a mixture of terpenoids, majorly monoterpenes (C10, about 90% EO content) and sesquiterpenes (C15), but they may contain diterpenes (C20) and numerous low-molecular-weight aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters, or lactones, as well as non-nitrogenous and sulphur-containing compounds [6,18,19].

Essential Oils Sources and Types
Essential oils (EO), recognzed as volatile oils, are commonly derived from edible, medicinal, herbal, or spice plants. The main plant tissues for EO deposition vary across the plants. They can be the leaves, flowers, stem, seeds, roots, rhizomes, or barks. The EO deposits are mostly extracted by using either steam distillation, hydro distillation, or organic solvent extractions [18]. The EO compounds are chemically a mixture of terpenoids, majorly monoterpenes (C10, about 90% EO content) and sesquiterpenes (C15), but they may contain diterpenes (C20) and numerous low-molecular-weight aliphatic hydrocarbons, acids, alcohols, aldehydes, acyclic esters, or lactones, as well as non-nitrogenous and sulphur-containing compounds [6,18,19]. Table 1. Major compounds of selected essential oils in different botanical fractions of various plants.
EOs can be obtained from not only the previously mentioned plant parts, but also other parts such as seeds, rhizomes, tree bark, tubers, and buds. Nigella sativa L. and Carum carvi L. are examples of medicinal plants where EOs are extracted from seeds as black cumin seed oil and caraway oil, respectively. The primary active compound in black cumin seed oil is para-cymene (37.3%) [22], while caraway oil contains carvone (76.8-80.5%) [23]. Other EOs such as turmeric oil, with 1,8-cineole (37.3%) being the main constituent, are taken from the rhizome Curcuma longa L. [25]. Cinnamon oil (E-cinnamaldehyde, 97.7%) is derived from the bark of the Cinnamomum zeylanicum tree [24], while clove oil (eugenol 88.6%) was extracted from the buds of Eugenia caryophyllata [26], and garlic oil (diallyl disulphide, 53%) was extracted from Allium sativum tubers. Table 2 reviews various research findings on the effects of EO, in the form of either extracts or whole plants, on ruminant fermentation profiles, gas (GP) and CH 4 productions, and animal performance and health. Patra and Yu [45] reported that various EO supplementations reduced degradability, GP, and CH 4 output, in line with decreasing archaea, protozoa, and cellulolytic bacteria. Protozoa and the majority of cellulolytic bacteria produce H 2 as their end product of fermentation, which is mainly utilized by methanogens (archaea) to form CH 4 in the rumen [46,47]. Lower CH 4 can be produced where more H 2 can be competitively converted, along with carbon dioxide (CO 2 ), to form acetate by hydrogenotrophic acetogens [48,49]. However, acetogens are able to utilize H 2 and CO 2 to produce acetate in the rumen, where methanogens are greatly inhibited [50]. If acetogenesis is dominant over methanogenesis, it can result in the predominant uses of H 2 and CO 2 by acetogens to produce acetate [48,51]. Reduced rumen CH 4 formation due to EO supplementations were also reported by other investigations [52,53].   In vivo dairy ewes Increased milk production (L/ewe/d), from 1.57 (control) to 1.68, 1.88, and 2.12 (50, 100, 150 mg EO/kg, respectively) but no effect on milk composition, as well as reduced urea concentration and somatic cell count at the greatest dose; no effect on cellulolytic bacteria and protozoa but decreased hyper-NH 3 -producing bacteria; no effect on pH; reduced NH 3

In vivo dairy cows
No effect on feed intake, Increased water intake for dose 48 mg/L; no effect on DM, OM, CP digestibility, milk production, and fat but increased protein in milk; no effect on pH and NH 3 but increased VFA for doses 16 and 32 mg/L; decreased A:P for 16 and 32 mg/L but increased A:P for 48 mg/L; no effect on total viable bacteria, cellulolytic, and protozoa counts for all doses of EO [58]  In vivo lactating ewes Thyme and celery increased weight gain and milk production. Thyme enhanced feed intake and nutrient digestibility. Thyme is preferred to celery in the diets of lactating ewes. [62] The use of EO as a dietary additive for ruminants in this article focuses on the research conducted in vivo. The in vitro research was still included, since it has been continued with the in vivo tests. It is intended that the utilization of EO as a dietary additive has confirmed its effect on livestock directly. Several parameters discussed in this article are related to the effect of EO on in vivo ruminant performances, for example, feed intake, body weight gain, feed efficiency, and nutrient digestibility. If the research was preceded by an in vitro test, the parameters to be observed were in vitro dry matter (IVDMD) and organic matter (IVOMD) digestibility, volatile fatty acids (VFA), ammonia (NH 3 ), and CH 4 outputs.
An in vivo experiment to observe the effect of EO on ruminants was carried out to investigate the use of a more mixed form of EOs compared to a single form. Geraci et al. [55] investigated a mixture of cinnamaldehyde and eugenol with a total administration of 400 mg/steer mixed into the mineral mixture. The same mixture with different concentrations was also tested by Tager and Krause [57] in dairy cows. The EO blend used by Geraci et al. [55] and Tager and Krause [57] reported no effect on DMI and VFA profiles in both feedlot cattle and dairy cows, respectively. A mixture of EO consisting of thymol, eugenol, vanillin, guaiacol, and limonene (Crina Ruminants, Switzerland) at 50, 100, and 150 mg/kg DM, respectively, given to dairy ewes, showed an improvement in milk production, but it had no impact on the milk compositions [56]. Soltan et al. [58] also reported insignificant effects of EO supplements containing eucalyptus oil, menthol, and mint on feed intake, DM, OM, CP digestibility, and milk production, except for enhanced milk protein in dairy cows.
Chaves et al. [59] observed that cinnamaldehyde and juniper berry EO additions in the diet increased average daily gain (ADG) but other studies reported that cinnamaldehyde, carvacrol [60], and oregano [61] EO supplementations had no effect on ADG in growing lambs.
It was reported that EO additions in the diet of growing lambs had no impact on carcass weight, meat yield [59][60][61], sensory parameters [60], tenderness [61], meat flavor, or overall fatty acid compositions [59]. However, Simitzis et al. [61] observed increased pH and the color of meat lambs as the result of EO supplementation, and a decrease in lipid oxidation during refrigeration and long-term freezing.
Research using EO mixture showed that the obtained effect was not considerably significant, and it was difficult to define which EO had the strongest influence. By using the same EO mixture, the effect of different inclusion levels can also have different impacts on livestock. Thus, this needs to be studied more deeply by considering the role of each EO containing different chemical substances.
It seems that the use of EOs as dietary additives to mitigate CH 4 output by the rumen in in vitro evaluations is nearly conclusive. However, the results of the effects of various EO inclusions into different ruminant diets on GP, VFA profiles, NH 3 , pH, and feed degradability parameters are still inconsistent. This is understandable, since there are naturally many sources of EO, and each of them may have different chemical constituents, so that the interaction among the chemical components of EO, doses, nutrient characteristics of different diets, and existing microbial populations in the rumen needs to be appropriately understood when planning similar research in the future. Table 3 summarizes the results of several studies using EO as anthelmintics for ruminants. The EO supplementation is also beneficial in improving animal health by combating parasites. Adding both Eucalyptus staigeriana [63] and Lippia sidoides [64] EOs in the diets of goats and sheep, respectively, was effective in helping animals against gastrointestinal nematodes, such as Haemonchus spp. and Trichostrongylus spp.  Citrus sinensis was more effective on eggs but Melaleuca quinquenervia was twice more effective on larvae.

Effect of Essential Oils as Anthelmintics for Ruminants
[71] Research on the effect of EO on reducing parasites and improving ruminant health was also carried out using an in vitro method. These in vitro experiments have been done to examine the presence of anthelmintic activities of various types of EO. The researchers have used different parasites in various growth phases such as eggs, larvae, and adult parasites using different methods of assessments. Most of the EO treatments indicated a reduction in the number of eggs and larvae of Haemonchus contortus [66,68,74]. Ferreira et al. [69] concluded that EOs from Thymus vulgaris could inhibit egg hatching, as well as the larval development and motility, of Haemonchus contortus in sheep.
Haemonchus contortus, Trichostrongylus spp., Fasciola hepatica, Rhipicephalus microplus, and Haemonchus polygyrus are widely studied as harmful parasites to ruminants. Several studies took EOs from various types of plant parts, especially those above the ground (not roots). Macedo et al. [63] conducted a study using EOs derived from Eucalyptus staigeriana in sheep infected with Haemonchus contortus. The results showed that EOs from Eucalyptus staigeriana was able to reduce worm eggs and larval development, and combat nematodes in the digestive tract of sheep. Similarly, a study performed by Camurça-Vasconcelos et al. [64] confirmed that EOs from Lippia sidoides increased the ability to combat nematodes such as Haemonchus contortus and Trichostrongylus spp. in sheep. Additionally, the inclusion of about 3% flaxseed oil in the diet of sheep could reduce the number of fecal egg counts [65].
An anthelmintic effect was also shown by EOs derived from the flowers and leaves of Ruta chalapensis [72]. The EO was tested in vitro using Haemonchus contortus derived from goats, and compared with albendazole. The results showed that EOs from leaves gave a higher inhibitory impact on worm hatching than EOs from flowers. Meanwhile, EOs derived from flowers showed an inhibition of motility of up to 87.5% after 8 h of exposure.
As mentioned earlier, the anthelmintic test of medicinal plants can use various types of methods. A study conducted by Katiki et al. [73] evaluated the anthelmintic Cymbopogon schoenanthus against Trichostrogylus spp. by using different methods, namely, the egg hatch assay (EHA), larval development assay (LDA), larval feeding inhibition assay (LFIA), and larval ex-sheathment assay (LEA). All of these methods validated that Cymbopogon schoenanthus potential as an anthelmintic, although it had to be retested in vivo. Similarly, Zanthoxylum simulans' EO has been tested in vitro using the EHA, LDA, and larval migration inhibition assay (LMIA), which confirmed that this EO had an anthelmintic potential to inhibit larval development of Haemonchus contortus in sheep [70].
The effects of EOs as anthelmintics are related to the interaction of these compounds with the structure of the parasite. This occurs when the lipophilic compounds, such as essential oil constituents, can break or damage the cell membrane of the parasite, thus affecting membrane permeability and leading to some enzyme and nutrient losses [74]. It is also possible that these Eos inhibit cell growth and differentiation, a very rapid process of worm egg embryogenesis [71].

Polyphenol Sources, Types and Uses
Polyphenols, such as tannins, are plant bioactive substances with various molecular weights and complexities. These compounds can bind to dietary proteins in aqueous solutions [75,76]. Although some pure plant polyphenols may be rarely soluble in water, their natural interactions ensure that some of those can be soluble in aqueous media [77]. Tannins contain multiple phenolic hydroxyl units that are able to configure complexes majorly with proteins, and minorly with metal ions, amino acids and polysaccharides [75]. Broadly, tannins are divided into two major groups: hydrolysable and condensed tannins (CT).
Hydrolysable tannins, known as gallotannins and ellagitannins, contain a structure based on a gallic acid unit. These are commonly identified as polyesters with D-glucose (gallotannins), while derivatives of hydroxydiphenic acid (ellagitannins) are developed from the oxidative coupling of contiguous gallolyl ester groups in a polygallolyl D-glucose ester [78]. Haslam [78] illustrated two pathways of gallic acid biosynthesis: (a) direct dehydrogenation of an intermediate in the shikimate pathway, as well as the retention of oxygen atoms of the alicyclic precursor, (b) a derivative of the end-product of the pathways.
Tannins contained in plants can be found in all parts of the plant, such as in sainfoin (Onobrychis viciifolia), with the largest content of Quercetin 3-rutinoside (6.15 mg/g DM) [79]. In addition to the plant as a whole, tannins are also found in leaves, young leaves, tree stalks, tree bark, core wood, and fruits. Leaves of Camellia sinensis (green tea), Pistachia lentiscus, and Phillyrea latifolia are known to contain tannins, where their dominant tannin contents are epigallocatechin gallate (94.6 mg/g DM) [7], cholorogenic acid (17.4 mg/L), and oleuropein (167.0 mg/L) [80], respectively. Several other plants containing tannins are described in Table 4.

Effect of Tannins as Feed Additives on Ruminants
Tannins reduce the solubility and rumen degradability of most dietary proteins, due to their ability to bind proteins. As a consequence, they may decrease the rumen NH 3 output and enhance the protein availability and non-NH 3 -N supply to be absorbed in the small intestine [6,14,76]. Even though NH 3 is a main source of N for rumen microbes, its fast or over production can exceed the ability of microbes to use it. This may result in an excessive NH 3 supply that, after absorption via rumen wall, can enter the blood stream, liver, and finally be excreted in urine as an N waste, causing potential risks for the environment [81,82]. Reduced CH 4 by 14-29% but decreased DMI and milk yield (especially in TAN-2); TAN-2 decreased fat (19%) and protein (7%) contents in the milk; no effect on protein and lactose contents; decreased digestible energy and N lost in urine [89]   In vivo dairy cows Decreased pH, NH 3 , calculated CH 4 , protozoa; no effect on tVFA but reduced A:P; no effect on milk yield, fats, CP, lactose, and energy contents in milk, DM, OM, and NDF digestibility [95] 13.

In vivo lambs
Recommendation of using Accasia mearnsii in lamb diet up to 40 g CT/kg DM, due to increased nutrient intake, digestibility, growth performance and feed efficiency. [97] The impacts of tannins as natural additives in various diets of ruminant have been studied using different in vivo, in vitro, and in sacco methods. Guglielmelli et al. [86] found that adding Sainfoin hay into a diet of cows gave a lower in vitro NH 3 production than alfalfa hay as the low tannins' counterpart. Quebracho extract addition into a diet of sheep wethers resulted in a lower ruminal NH 3 and blood urea N concentrations [94]. Adding tannin extract from Vaccinium vitis-idaea into a diet of dairy cows decreased NH 3 production [95]. Grainger et al. [89] concluded that tannin extracts from Acacia mearnsii barks in a diet of dairy cows reduced urinary N loss. A similar decrease in urinary N excretion was reported in wethers supplemented by a tannin extract from Acacia mearnsii [98]. Nevertheless, Puchala et al. [99] reported that there was no difference for NH 3 productions between goats fed fresh Sericea lespedeza, rich in tannins, and those fed either alfalfa or sorghum-Sudan grass. A study comparing the growth stages of purple prairie clover, between vegetative and flowering stages with different CT contents, showed that they were not different in in vitro rumen NH 3 production [84].
Tannins can also decrease rumen CH 4 output by reducing the inter-species transfer of H 2 into methanogenic bacteria, and hence depressing their growth [6,76,85]. Huang et al. [85] informed that CT extract supplementation from Leucaena leucephala reduced in vitro rumen GP and CH 4 releases. Moreover, tannin extract addition from Acacia mearnsii into a diet of dairy cows reduced CH 4 production [89]. It was similarly reported that goats fed either fresh Sericea lespedeza, rich in tannins, or its hay produced less CH 4 in comparison with those fed either alfalfa or sorghum-Sudan grass [99]. However, Guglielmelli et al. [86] reported that Sainfoin hay released higher in vitro CH 4 from the rumen than alfalfa hay.
Sainfoin hay supplementation produced higher rumen in vitro VFA and acetate, but no difference was reported in the acetate:propionate (A:P) ratio compared with alfalfa hay [86]. Wood et al. [83] found that Chrysanthemun coronarium supplementation likely acted to increase acetate but reduce propionate. Nonetheless, Cieslak et al. [95] reported that adding tannin extracts from Vaccinium vitis-idaea in a diet of dairy cow had no effect on VFA, but reduced the A:P ratio in the rumen fluid.
It was reported that CT extract supplementation from Leucaena leucephala had no impact on IVDMD, except for it being lower for the high dose [85]. An in vitro experiment comparing the growth stage of purple prairie clover between vegetative and flowering stages (58.6 and 94.0 g CT/kg DM, respectively) indicated that the vegetative stage had a higher IVDMD than flowering stage [84]. An in sacco investigation by Azuhnwi et al. [87] found that adding condensed tannins from sainfoin (Onobrychis viciifolia Scob) into a diet of dairy cow reduced DMI and CP degradability. Meanwhile, Guglielmelli et al. [86] reported that Sainfoin hay resulted in greater IVOMD than that by alfalfa hay.
Kozloski et al. [98] indicated that adding tannin extract from Acacia mearns to a diet of sheep wethers resulted in a lower DMI and the digestibility of DM, OM, neutral detergent fiber (NDF), and N. Grainger et al. [89] also showed a reduction in DMI and milk yield in dairy cows supplemented with tannins extracted from Acacia mearnsii. Different things were presented by Costa et al. [97], in which the addition of Acacia mearnsii up to 40 g CT/kg (Acacia mearnsii contains 700 g CT/kg) in the lamb feed could increase nutrient intake and digestibility, as well as increase growth and feed efficiency. However, Briceño-Poot et al. [88] reported that the addition of Acacia pennatula or Enterolobium cyclocarpum into a diet of sheep resulted in a higher DMI, especially for those supplemented with Acacia pennatula. Similarly, it was reported that goats fed either fresh Sericea lespedeza or its hay had higher DMI but lower DM and N digestibility in comparison with those fed either alfalfa or sorghum-Sudan grass [99]. Owens et al. [93] informed that adding quebracho tannin extract from Aspidosperma quebracho into a diet of lambs resulted in no impact on DMI, digested DM, digested energy, or digested NDF, but increased N digestibility. Galicia-Aguilar et al. [100] reported that sheep supplemented by Havardia albicans had a similar DMI but lower DM digestibility. Cieslak et al. [95] observed that adding tannin extract from Vaccinium vitisidaea into a diet of dairy cows had no impact on milk production and its fat, CP, lactose, and energy contents, as well as DM, OM, and NDF digestibility. In addition, adding quebracho tannins extract into a diet of sheep increased cis9, trans11 CLA (conjugated linoleic acid, rumenic acid) and polyunsaturated fatty acids (PUFA), but reduced saturated fatty acids (SFA) in the longissimus muscle [92] and increased vaccenic acid (trans11 C18:1) with no effect on stearic acid (C18:0) compositions in the rumen fluid [91].
Tannin addition into ruminant diets increased the rumenic acid and PUFA and decreased SFA in ruminant products, such as milk and meat, via modified bio-hydrogenation by altering the rumen microbial population [83,91,92]. Tannin supplementation, however, is thought to be associated with reduced feed intake, resulting in possible reduced nutrient intakes, digestibility, animal performance. These responses may be due to the possible toxicity of tannin-containing diets to animals [76,101].

Effect of Tannins as Anthelmintics on Ruminants
Azaizeh et al. [80] reported that the Pistachia lentiscus and Phillyrea latifolia extracts inhibited the exsheathment of gastro-intestinal nematode larvae in vitro, while sheep supplemented with Havardia albicans had less Haemonchus contortus in their faeces [100]. Julaeha et al., [3] found that adding Jatropha multifida leaves into a diet of lambs reduced Trichostrongylus spp. fecal eggs counts. Tannins have the potential to increase animal health via their antioxidant properties and to prevent bloat as well as to break protein-rich cells of nematodes [102].
The other ruminant studies in vivo showed that tannins had the anthelmintic potentials. Saratsi et al. [103] stated that Ceratonia siliqua, rich in CT, had an anthelmintic effect. The cashew apple fiber added into a sheep's diet as a source of tannins showed 40.8% effectiveness as an anthelmintic compared to a monepantel anthelmintic [104]. The other herbal plants tested in vivo such as green tea, oak leaves, and mixed herbs showed their effects on increasing host resistance to parasites, reducing the number of parasites, and increasing livestock productivities [105][106][107].  Acevedo-Ramírez et al. [108] conducted a sheep in vitro study using tannins derived from a chestnut tree. The results indicated that tannins can cause the death of adult Haemonchus contortus, so that tannins can be used as an alternative to conventional nematode control agents in ruminants. This is similar to the results reported by Mazhangara et al. [109], who tested tannins in Elephantorrhiza elephantine. Studying tannins as anthelmintics was also carried out using the larval ex-sheathment inhibition assay (LEIA), where Pistacia lentiscus, Phillyrea latifolia, and Inula viscosa, harvested in different seasons, showed different anthelmintic effectiveness. Azaizeh et al. [110] and Santhi et al. [111] tested an ethanol extract of Inula viscosa, Salix alba, and Quercus calliprinos using an in vitro developmental assay, which showed that these tannin-rich plant extracts were considerably toxic to the eggs and larvae of Heterorhabditis bacteriophora.
Tannins can act as an antiparasitic agents in ruminants. The efficacy of tannins in reducing gastrointestinal nematodes is by increasing the host response to parasites. The capability of tannins to bind to proteins is able to protect them from rumen degradation, and improve protein flow and amino acid absorption in the small intestine [3]. Increased protein supply in the small intestine is seen to enhance host homeostasis and immune response to helminths [12].

Saponin Sources, Types, and Uses
Saponins are distributed in most parts of the plant, such as the leaves, seeds, roots, tubers, and tree bark. Some plant sources that contain tannins are Camelia sinensis var. Assamica, Dioscorea pseudojaponica Yamamoto, and Quillaja saponica. All of these plants have saponins in various forms, as described in more details in Table 2. Saponins are a diverse unit of low-molecular-weight, plant-bioactive compounds. Saponins have the capability to form stable soap-like foams in watery solution. Table 7. Chemical characteristics of saponins in different botanical parts of some saponin-rich plants.

Plants Scientific Names Main Parts Major Bio-Active Compounds References
Chinese chive Allium tuberosum

Plants Scientific Names Main Parts Major Bio-Active Compounds References
Yam [ 15] Chemically, saponins comprise a sugar moiety, commonly containing glucose, galactose, glucuronic acid, xylose, rhamnose, or methyl pentose, which is glycosidically related to a hydrophobic aglycone (sapogenin) in the form of either triterpenoids or steroids [5,115]. Triterpenoids are widely distributed in nature in comparison with steroids [116]. The usual form of triterpenoid aglycone is a derivative of oleanane, while the main forms of steroid aglycones are mostly found in the spirostanol and furostanol derivatives [115,116]. The aglycone may consist of one or more unsaturated C-C bonds [5]. The chain of oligosaccharides is commonly attached at the C3 location (monodesmosidic), but there are numerous saponins found to have an extra sugar moiety at the C26 or C28 positions (bidesmosidic) [116]. Wina et al. [115] also reported that there were two general types of triterpenoid saponins: neutral and acidic. Neutral saponins have their sugar components attached to sapogenin, while acidic saponins have their sugars moiety containing uronic acid, or with one or more carboxylic units attached to the sapogenin [115].

Effects of Saponins as Dietary Additives on Ruminants
Several studies have shown that tea saponins have a suppressing impact on the release of CH 4 and NH 3 in vitro [117] and in vivo by using growing lambs [118]. The CH 4 reduction was supported by the reduction in protozoa and particularly the protozoarelated methanogens [115,119]. Saponins can act as defaunation agents via a sterol-saponin interaction in the protozoal cell membrane, hence affecting the methanogenic protozoa [115]. Since protozoa can be a predator for bacteria, at an appropriate level, defaunation may improve the population of bacteria and may increase N utilization, leading to improved animal growth and meat or milk productions [115]. Less protozoa in the rumen is also likely to result in less acetate production, since most fermentation end products of protozoa comprise acetate [6,115].

In vivo goats
No effect on DM, N, or ADF intakes; no effect on DM, N, or ADF digestibility, either in rumen or small intestines; no effect on amino acid digestibility in small intestine; no effect on rumen pH, VFA, A:P, or NH 3 [124] 6.

In vivo lambs
No effect on feed intake and daily gain; reduced CH 4 (L/kg DMI); increased tVFA but no effect on A:P; decreased ruminal pH and reduced NH 3 ; no effect on methanogens, fungi, R. flavefaciens, orand F. succinogenes, but decreased protozoa populations. Reduced SFA, cis9, trans11 CLA/vaccenic acid ratio; increased MUFA, but no effect on PUFA (longissimus dorsi muscle) [118,126]

In vivo lambs
No effect on the intakes of DM, OM, CP, or NDF, or the digestibility of DM, OM, or CP, but decreased NDF digestibility; no effect on N balance, N supply, pH, or NH 3 but decreased protozoa numbers and glucose; no effect on ADG, cooking loss, or meat pH (24 h post mortem), but decreased carcass weight [127] Reduced the concentration of cis9 C14:1 (longissimus dorsi muscle) and its desaturation index; 12 mg had higher C20:4n6 than control and 6 mg; 12 mg had lower α-linolenic:linoleic ratio than control; no effect on muscle cholesterol levels [10] 10.

In vivo goats
Acacia concinna had no influence on FA composition in muscle and adipose tissues. Syzygium aromaticum has the potential to enhance the health-promoting VA and cis-9, trans-11 CLA concentrations in the meat of goats [128] 11.
Goel and Makkar [130] reported that adding saponin extract reduced NH 3 production, but Istiqomah et al. [122] found in vitro that waru leaf supplementation had no effect on NH 3 production. Although Mao et al. [118] reported that adding tea saponin extract into a diet tended to reduce NH 3 production (143.0 vs. control, 167.5 mg/L), Zhou et al. [124] observed in vivo that green tea saponin extract additions at 0.4, 0.6, or 0.8 g saponins/kg dietary DM had no effect on the NH 3 production of goats. Similarly, Nasri et al. [127] found in vivo that adding saponins extract from Quillaja saponaria at 6, 12, or 18 mg sapogenin/kg dietary DM had no effect on the NH 3 production of the lambs.
It was reported in vitro that waru leaf inclusions into a Napier grass-based diet were likely to increase VFA, but Wang et al. [121] reported in vitro that saponin extract supplementation from Gynostemma pentaphyllum reduced VFA without affecting VFA proportions. Mao et al. [118] observed in vivo that adding tea saponin extract into a diet of lambs increased VFA with no effect on the A:P ratio, while Zhou et al. [124] observed that green tea saponin extract inclusions had no effect on either the tVFA or A:P ratio in the rumen liquid of goats.
Wang et al. [121] found in vitro that adding saponins extract from Gynostemma pentaphyllum increased ruminal pH, but Istiqomah et al. [122] found in vitro that waru leaf addition in Napier grass resulted in no impact on ruminal pH. An in vivo lamb study by Mao et al. [118] reported that adding tea saponin extract into a diet decreased ruminal pH, but Zhou et al. [124] observed in vivo that green tea saponin extract supplementation had no impact on ruminal pH in goats. Similarly, Nasri et al. [127] found in vivo that saponin extract supplementation from Quillaja saponaria into oat hay-and barley-based diets had no impact on ruminal pH in lambs.
It was observed in vitro that saponin extract inclusions from either Achyranthus aspara, Tribulus terrestris or Albizia lebbeck had no effect on IVDMD [130]. Meanwhile, an in vivo study by Nasri and Ben Salem [123] found that adding saponin extract from Agave Americana at 120, 240, or 360 mg saponins/kg and Quillaja saponaria at 120 mg saponins/kg dietary DM had no effect on DMI and nutrient intakes, and no effect on the OM, CP, and NDF digestibility of lambs. Similarly, Owens et al. [93] reported that adding saponin extracts from Quillaja saponaria at 20 g saponins/kg (Beet pulp-based diet containing alkaloids, either gramine at 2 g/kg or methoxy-N, N-dimethyltryptamine at 0.03 g/kg diet) had no effect on DMI or the total digested DM, energy, N, and NDF by lambs. Mao et al. [118] studied in vivo that tea saponin extract inclusions had no impact on feed intakes and weight gain of lambs. Zhou et al. [124] studied in vivo that green tea saponin extract supplementation had no impact on the intakes and the digestibility of DM, N, and ADF of goats. Li and Powers [125] added either Yucca schidigera (YS), Quillaja saponaria (QS) or Camellia sinensis extracts (TS, tea saponins) into a corn-and corn-silage-based diet of steers, and found that QS and YS had no difference compared with the control diet in DMI and ADG, but the N intake of YS was lower than the control diet and QS, while TS had higher DMI and N intake but having a similar ADG to the control diet. In addition, it was reported in vivo that adding saponin extracts from Quillaja saponaria at 6, 12, and 18 mg sapogenin/kg DMI in an oat hay-and barley-based diet had no effect on the intakes of DM, OM, CP, and NDF, the digestibility of DM, OM, and CP, as well as ADG, cooking loss, and meat pH, but decreased NDF digestibility in lambs [127]. Brogna et al. [10] also found a reduction in the concentration of C14:1 cis-9 from the longissimus dorsi muscle and its desaturation index, increased C20:4n-6, and decreased α-linolenic:linoleic ratio at a saponin level of 12 mg, with no effect on muscle cholesterol concentrations of lambs. Meanwhile, Mao et al. [126] reported that adding tea saponin extracts (>60% triterpenoid saponins) into a diet of lambs reduced SFA and the rumenic:vaccenic acid ratio and increased MUFA, but it had no effect on PUFA in the longissimus dorsi muscle.
Another study was conducted in vitro by Mandal et al. [128] and Patra and Yu [45] on goats and cattle, respectively. Acacia concinna showed no impact on fatty acid conformation in muscle and adipose tissue, while Quillaja Saponaria showed its effect on decreasing CH 4 production. On the other hand, the use of saponins from tea leaves in vitro and in vivo in dairy cows has shown its effect on decreasing lactation performance and dry matter consumption, but did not reduce CH 4 production, so that saponins in tea are considered inefficient to reduce CH 4 emissions [129].

Effect of Saponin as Anthelmintics on Ruminants
Botura et al. [131] reported, in vivo, that supplementing either sisal waste extract (SWE) (Agave sisalana, containing hecogenin and tigogenin) at 1.7 g/goat/day or levamisole phosphate (LEP) (6.3 mg/kg) as a positive control into grass hay-fed goats reduced fecal egg counts by a maximum of 50.3% (SWE) and 93.6% (LEP). In this study, LEP reduced the recovered parasites from the digestive tract by 74%, but a small decrease of parasites was reported for SWE. There was no toxicity effect reported from both treatments, as measured by the histological analysis of the kidney and liver. Another experiment was carried out using the egg hatch assay (EHA) and larval migration inhibition (LMI) methods using Phytolacca icosandra [132] and Agave sisalana (aqueous extract) [133]. Both studies showed that Phytolacca icosandra in ethanol and dichloromethane extracts could destroy Haemonchus contortus eggs and larvae, while the saponins contained in Agave sisalina could also attack nematodes in the digestive tract of ruminant animals. Table 9. Effect of different saponins as possible anthelmintics on ruminants.

No
Saponins Test systems Outputs References

1.
Sisal waste extract (SWE) (Agave sisalana, containing saponins in the form of sapogenins hecogenin and tigogenin) at 1.7 g/goat/day; levamisole phosphate (LEP) (6.3 mg/kg) as a (+) control In vivo goats fed by grass hay Reduced fecal egg count by max. 50.3% (SWE) and 93.6% (LEP); LEP reduced the recovered parasites from the digestive tract by 74% but a low decrease of those parasites for SWE. No toxicity effect from both treatments assessed by histological analysis of the liver and kidney [131] 2.
Phytolacca icosandra (ethanol, n-hexane, and dichloromethane extract) at 0. In vitro LMI and EHA assays by a donor sheep with a monospecific infection of Haemonchus contortus Saponins were only found in the ethanolic extract of Phytolacca isocandra. Ethanolic and dichloromethane extracts of the plants showed in vitro anthelmintic activity against the H. contortus eggs and the L 3 larvae. However, the hexanic extract of the plant leaves failed to show any in vitro anthelmintic activity.
[132] The saponin fractions showed no ovicidal activity while flavonoid fractions did not show activity against larvae. Agave sisalana was active against the gastrointestinal nematodes of goats, related to the presence of homo-isoflavanoid saponin compounds. [133]

4.
Elephantorrhiza elephantina roots (83.28 ± 1.72% saponins) in ethanol, methanol, and water extract at 1.87, 3.75, 7.5, and 15 mg/mL In vitro adult motility inhibition assay with naturally infected goats Ethanol, methanol, and water extract of Elephantorrhiza elephantina roots showed a potential anthelmintic activity against adult Paramphistomum cervi worm motility, in botha a time-and dose-dependent mannerand. [109] The hatching process of nematode eggs begins with a stimulus from the environment, which causes the larvae to release several enzymes, such as proteases, lipases, and chitinases, that function to degrade the egg membrane [133]. The flavonoid compounds contained in Agave sisalana can inhibit the activity of these enzymes, so that changes in enzyme activity interfere with the egg-hatching process, resulting in the destruction of infectious worms [133].

Conclusions and Perspectives
Essential oils, polyphenols, and saponins are plant secondary metabolites found in various type of plants that can be extracted from different botanical parts of many plants. These materials can function as dietary additives and anthelmintics to increase the production and health performance of ruminants. Each bioactive constituent has a specific function and efficacy to achieve pre-defined objectives. However, the literature shows that these compounds may be variable in their effectiveness depending upon the plant sources, extraction methods, amounts, and diets in various studies in different situations. Several bioactive-compound-based dietary supplements can reduce methane or nematode parasites. However, these great reductions are sometimes followed by significant declines in feed intake and performance of the animals. Therefore, it is essential to select the most appropriate plants that contain compounds selected for their appropriate dosages and applications, to either optimize rumen function or reduce methane and anthelmintics in a range of ruminant animals. Moreover, it is vital to test the potential safety, affordability and efficiency of their dietary inclusion for ruminant animals, and, consequently, the impacts on consumers, and the environment.

Data Availability Statement:
No new data were created or analyzed in this study. Data sharing is not applicable to this article.